Abstract
The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common beta (betac) receptor is shared by the three cytokines and functions together with cytokine-specific alpha subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human betac (hbetac) and unidentified cysteine residues in the N-terminal domains of the alpha receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hbetac is part of the cytokine binding epitope of this receptor and that an IL-3Ralpha isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hbetac and the related mouse receptor, beta(IL-3), could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hbetac led to both a complete loss of high affinity binding with the human IL-3Ralpha SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, beta(IL-3), not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Ralpha SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hbetac and beta(IL-3) in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.
Highlights
T-cells during immune responses and play key roles in the regulation of inducible hematopoiesis and inflammation
These studies illustrate that details of the mechanisms underlying activation by IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors are still emerging, and recent structural and biochemical studies suggest that receptor activation is likely to differ markedly from the paradigms established for other class I cytokine receptors
The D-E loop disulfide is well conserved in hc, mc, and IL-3 and is additional to the two structural disulfides present in the cytokine receptor homology module (CRM) of these receptors (Fig. 1D)
Summary
Cells and DNA Transfections—COS7 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. CTLL2 cell lines stably expressing wild type or mutant hIL-5R␣, mIL-3R␣ SP1, mIL-3R␣ SP2, hGM-CSFR␣, hIL-3R␣ SP1, or hIL-3R␣ SP2 and wild type or mutant hc or IL-3 subunits were generated in two steps with DNA introduced by electroporation, essentially as described previously [34]. Proliferation Assays—[3H]Thymidine incorporation assays were performed as described previously [34] to determine the capacity of the wild type or mutant receptor subunits to deliver a proliferative signal in CTLL2 stable cell lines in response to ligands. In order to assess the capacity of IL-3 subunits to bind mIL-3 directly, cold saturation assays were performed on COS7 cells transfected with cDNAs encoding wild type or mutant IL-3 subunits in the absence of the mIL-3R␣ cDNA, as described previously [25]. Scatchard plots were used for visual evaluation of the quality of the data and their fits to one site and two-site models
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
